The In Vitro Replication Cycle of Achromobacter xylosoxidans and Identification of Virulence Genes Associated with Cytotoxicity in Macrophages

ABSTRACT Achromobacter xylosoxidans is an opportunistic pathogen implicated in a wide variety of human infections including the ability to colonize the lungs of cystic fibrosis (CF) patients. The role of A. xylosoxidans in human pathology remains controversial due to the lack of optimized in vitro and in vivo model systems to identify and test bacterial gene products that promote a pathological response. We have previously identified macrophages as a target host cell for A. xylosoxidans-induced cytotoxicity. By optimizing our macrophage infection model, we determined that A. xylosoxidans enters macrophages and can reside within a membrane bound vacuole for extended periods of time. Intracellular replication appears limited with cellular lysis preceding an enhanced, mainly extracellular replication cycle. Using our optimized in vitro model system along with transposon mutagenesis, we identified 163 genes that contribute to macrophage cytotoxicity. From this list, we characterized a giant RTX adhesin encoded downstream of a type one secretion system (T1SS) that mediates bacterial binding and entry into host macrophages, an important first step toward cellular toxicity and inflammation. The RTX adhesin is encoded by other human isolates and is recognized by antibodies present in serum isolated from CF patients colonized by A. xylosoxidans, indicating this virulence factor is produced and deployed in vivo. This study represents the first characterization of A. xylosoxidans replication during infection and identifies a variety of genes that may be linked to virulence and human pathology. IMPORTANCE Patients affected by CF develop chronic bacterial infections characterized by inflammatory exacerbations and tissue damage. Advancements in sequencing technologies have broadened the list of opportunistic pathogens colonizing the CF lung. A. xylosoxidans is increasingly recognized as an opportunistic pathogen in CF, yet our understanding of the bacterium as a contributor to human disease is limited. Genomic studies have identified potential virulence determinants in A. xylosoxidans isolates, but few have been mechanistically studied. Using our optimized in vitro cell model, we identified and characterized a bacterial adhesin that mediates binding and uptake by host macrophages leading to cytotoxicity. A subset of serum samples from CF patients contains antibodies that recognize the RTX adhesion, suggesting, for the first time, that this virulence determinant is produced in vivo. This work furthers our understanding of A. xylosoxidans virulence factors at a mechanistic level.

. Determined domain architecture of ArtA in 33 Ax genomes with respect to the GN050 RTX adhesin. Using an anvi'o pipeline, 8 genomes were found to encode a complete ArtA while 25 other sequences in the gene cluster encode the highly conserved CT including vWA, RTX repeats and T1SS signal sequence. In genomes containing an ArtA CT but lacking further annotation, manual determination of the VCBS repeating units were determined and denoted as "determined." R; retention module. VCBS; repeating units. vWA; von Willebrand type A. RTX; Ca 2+ binding RTX repeats. T1S; T1SS signal sequence. Figure S4. Pangenomic analysis of 88 Ax genomes with respect to GN050 synteny. Genome layers are ordered by the maximum likelihood relationship of 77 single-copy core genes. Displayed is the presence or absence of gene clusters of homologous genes based on amino acid sequence identity. By this method, genomes that contained the artA gene cluster are shown in pink and genomes that lacked the artA gene cluster are shown in black. Minimum requirement for presence of artA in a genome included annotation of a vWA domain, RTX-repeats and a T1SS signal sequence ( Figure  S3). Figure S5. CF serum antibodies that recognize ArtA also recognize a HMW product produced by other Ax CF isolates. (A) Precipitated supernatants from overnight cultures of GN050::26F2, WT GN050, or Ax CF isolates Ax3, Ax4, Ax12, GN008, Ax5, AX13, AX14. (B) Western blot using CF serum ID: 9429 that was previously found to have GN050 ArtA reactivity. Reactivity of the serum against HMW bands was found in CF isolates Ax3, Ax4, Ax12, and GN008. Detection of a HMW similar to ArtA is denoted by red arrows.  Movie S1. Live imaging of J774a.1 cells infected with GN050 pDBD2sfGFP at an MOI:5 for 30 min followed by treatment with antibiotics to select for intracellular bacteria. After 30 min of treatment, the infection was replaced with antibiotic-free medium containing 0.36 μl/ml PI and imaged every 15 min for 20 h. Chloramphenicol (10 μg/ml) was supplemented into the infection to promote plasmid maintenance and sfGFP expression. Scale = 25 μm.
Movie S2. FITC and dsRED channels extracted from supplemental movie 1 to emphasize bacterial proliferation in relation to cytotoxicity. Scale = 25 μm.
Movie S3. Live imaging of J774a.1 cells infected with GN050 pDBD2sfGFP at an MOI:5 for 30 min followed by treatment with antibiotics to select for intracellular bacteria. After 30 min of treatment, the infection medium was replaced with medium containing 50 μg/ml gentamicin, 10 μg/ml polymyxin B, 0.36 μl/ml PI and imaged every 15 m for 20 h. Chloramphenicol (10 μg/ml) was supplemented into the infection to promote plasmid maintenance and sfGFP expression. Scale = 25 μm.
Movie S4. FITC and dsRED channels extracted from supplemental movie 2 to emphasize bacterial proliferation in relation to cytotoxicity. Scale = 25 μm.

Construction of pDBD2sfGFP and Modification of himar1
Antibiotic resistance is problematic in Achromobacter species. Initial characterization of antibiograms suggested that chloramphenicol could be useful at high concentrations (100-200 µg/ml) for selection in several clinical isolates from the Medical College campus, including GN050. As IncP plasmids appear to replicate in Achromobacter (4,5), we constructed pDBD2 utilizing the broad host range vector pCM62 (2). The CmΩ (6)

Transposon mutagenesis screen
To identify GN050 himar1 insertion mutants that were unable to induce cytotoxicity in J774a. were washed with HBSS (Gibco) and stained with 3% crystal violet in 5% methanol 5% ethanol 5% isopropanol for 5 min at room temperature (RT). Each well was then washed with deionized water to remove excess crystal violet followed by an image capture to document the staining pattern of each well.
Wells that retained a majority of crystal violet staining were thought to be infected with a GN050 mutant that was non-toxic to J774a.1 cells. These selected wells were paired with mirrored replica plate mutants that grew on medium containing 100 µg/ml chloramphenicol, confirming the presence of the selectable marker. Each selected GN050 mutant was struck on LB for further analysis. Mutants that grew in a comparable manner to WT GN050 on LB were processed in a secondary screen with a standardized MOI. Here, J774a.1 cells seeded at 1.75 x 10 5 cells per well in 24-well plates were infected with WT GN050 and mutant derivatives at an MOI of 10:1 for 6-8 h after a centrifugation of 600 x g for 5 min. To quantify cytotoxicity, adenylate kinase (AK) release assays were carried out as previously described (1). Genome location of himar1 insertions were determined for GN050 mutants that were at 70% or below of WT toxicity in the secondary screen.

Sequencing and functional annotation of himar1 insertion mutants
Clones, possessing similar growth properties to GN050 but defective for cytotoxicity in primary and secondary screens, were processed for genomic DNA isolation using the DNeasy ultraclean microbial kit according to manufacturers' instructions (Qiagen). One microgram of genomic DNA was digested with NotI in a 20 µl reaction. Restriction endonuclease reactions were heated to 65˚C for 20 min to inactivate NotI and 50 ng of DNA was ligated to rescue plasmid containing the R6Kori from himar1. Ligations were transformed into DH5a lpir and selected on medium with 30 µg/ml chloramphenicol. The resulting colonies were screened for kanamycin resistance. Plasmid DNA was isolated from colonies expressing both chloramphenicol and kanamycin resistance and subjected to DNA sequence analysis utilizing primers that bind to each end of himar1.

Quantitative reverse transcription-PCR (RT-qPCR)
GN050 and derived strains were grown in LB to an OD600 of 0. Primers and template were mixed with nuclease-free water and 10 µl of 2X SsoAdvanced Universal SYBR green Supermix (Bio-Rad). qPCR was performed on a CFX Connect real-time system (Bio-Rad) and analyzed using CFX Maestro software (v4.1).

Functional annotation of insertion mutants
Transposon insertions that mapped to an ORF were assigned functional annotation identifiers. Ax GN050 genome containing ORF identifiers (FASTAS) were exported from the NCBI genome database (CP053617) and converted into an anvi'o (v7, "Hope")-useable contig database file (.db). Functional annotations were identified against COG and PFAM databases. Protein sequences (.fa) were exported from anvi'o and imported into GhostKOALA (Kanehisa Laboratories) for KEGG annotation.
To determine the phylogenetic relationship of the 88 Ax isolates, an anvi'o (v7, "Hope") workflow for phylogenomics was used. A new bin collection in 'anvi-display-pan' was generated to identify core single-copy genes (SCG's) across Ax genomes. Because the strains were highly similar, custom homogeneity indices (Min number of genomes gene cluster occurs = 88, Max number of genes from each genome = 1, Max functional homogeneity index = 0.95, Min geometric homogeneity index = 0.9) were applied to identify 77 SCG's Alignments from the pangenomic analysis described above and using the bin collection with 77 SCG's were exported (.fa) using 'anvi-get-sequences-forgene-clusters' with the command '-concatenate-gene-clusters' and '-max-num-genes-from-eachgenome = 1'. A phylogenetic tree (maximum-likelihood method) was generated using 'anvi-genphylogenomic-tree' and visualized in 'anvi-interactive'. The tree was exported into a Newick file (.nwk) manipulated for aesthetics using inkscape (v1.0).

Transmission election microscopy (TEM)
Cells in culture dishes were fixed in mixture of 2.5% glutaraldehyde + 2% paraformaldehyde in 100mM sodium cacodylate buffer pH 7.4 (8) for 1 h at RT. Following fixation the cells were washed 3x5 min in cacodylate buffer then post fixed in reduced 1% osmium tetroxide (9) for 2 h on ice. Cells were scraped and the suspension was centrifuged at 8,000 x g for 10 min. The cell pellet was kept intact during washing (3 x 5 min) with distilled water then dehydrated through a graded methanol series followed by 2 x 10 min washes in acetonitrile before infiltrating with EMBed 812 epoxy resin and polymerization overnight at 70ºC. Ultrathin sections (70 nm) were cut, stained with uranyl acetate and lead citrate, and examined in a JEOL 1400 transmission electron microscope. Images were recorded using an AMT "nanosprint 12" digital camera.

Western blotting analyses
To prepare bacterial supernatants, GN050 and derived strains were grown in Tryptic Soy Broth (TSB) dialysate medium overnight at 37ºC shaking at 250 rotations per minute (rpm). Cultures were subject to centrifugation at 3,823 x g for 10 min at RT. Supernatants were harvested and filter sterilized through a 0.2 µm filter. Approximately 9.6 ml of supernatant was mixed with 12 ml of saturated ammonium sulfate solution (Thermo Scientific) and placed on ice for 3 h. Precipitated material was collected by centrifugation at 31,209 x g for 30 min at 4ºC followed by another brief centrifugation (31,209 x g for 5 min at 4ºC) to remove as much supernatant as possible. Pellets were suspended in 240 µl 1x SDS loading buffer (0.1 M dithiothreitol) and boiled for 5 min before loading on 8% acrylamide, SDS gels.

Figures and statistical analyses
Figures were generated and statistical analyses were performed using python (v3.8.5) in a JupyterLab notebook environment and figures were modified using Inkscape software (v1.1). When applicable, one-way or two-way ANOVA statistical tests were performed in combination with Tukey's post hoc analysis. Generally, cutoff for significance was at least p<.001 unless otherwise specified.
For each method, the statistical analysis used is indicated in the figure legend.